IDENTIFICATION OF DNA RECOGNITION SEQUENCES AND PROTEIN-INTERACTION DOMAINS OF THE MULTIPLE-ZN-FINGER PROTEIN ROAZ

Roaz, a rat C2H2 zinc finger protein, plays a role in the regulation o f olfactory neuronal differentiation through its interaction with the Olf-1/EBF transcription factor family. An additional role for the Roaz /Olf-1/ EBF heterodimeric protein is suggested by its ability to regul ate gene activation at a distinct promoter lacking Olf-1/EBF-binding s ites. Using an in vitro binding-site selection assay (Selex), we demon strate that Roaz protein binds to novel inverted perfect or imperfect repeats of GCACCC separated by 2 bp. We show that Roaz is capable of b inding to a canonical consensus recognition sequence with high affinit y (K-d = 3 nM). Analysis of the structural requirement for protein dim erization and DNA binding by Roaz reveals the role of specific zinc fi nger motifs in the Roaz protein for homodimerization and heterodimeriz ation with the Olf-1/EBF transcription factor, The DNA-binding domain of Roaz is mapped to the N-terminal 277 amino acids, containing the fi rst seven zinc finger motifs, which confers weak monomeric binding to a single half site and a stronger dimeric binding to the inverted repe at in a binding-site-dependent manner. Full-length protein can form di mers on both the inverted repeat and direct repeat but not on a single half site. These findings support the role of the TFIIIA-type Zn fing ers in both protein-protein interaction and protein-DNA interaction an d suggest distinct functions for specific motifs in proteins with a la rge number of zinc finger structures.